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Abstract

Background

Fourier Transform Infrared Imaging (FTIRI) is used to investigate the amide anisotropies
at different surfaces of a three-dimensional cartilage or tendon block. With the change
in the polarization state of the incident infrared light, the resulting anisotropic
behavior of the tissue structure is described here.

Methods

Thin sections (6 μm thick) were obtained from three different surfaces of the canine
tissue blocks and imaged at 6.25 μm pixel resolution. For each section, infrared imaging
experiments were repeated thirteen times with the identical parameters except a 15°
increment of the analyzer's angle in the 0° – 180° angular space. The anisotropies
of amide I and amide II components were studied in order to probe the orientation
of the collagen fibrils at different tissue surfaces.

Results

For tendon, the anisotropy of amide I and amide II components in parallel sections
is comparable to that of regular sections; and tendon's cross sections show distinct,
but weak anisotropic behavior for both the amide components. For articular cartilage,
parallel sections in the superficial zone have the expected infrared anisotropy that
is consistent with that of regular sections. The parallel sections in the radial zone,
however, have a nearly isotropic amide II absorption and a distinct amide I anisotropy.

Conclusion

From the inconsistency in amide anisotropy between superficial to radial zone in parallel
section results, a schematic model is used to explain the origins of these amide anisotropies
in cartilage and tendon.

Background

Tendon is a soft connective tissue that lies in between bones and muscles in animal
and human body to transfer the force experienced by muscle to the bone. Tendon therefore
has the nature to resist mechanical tension. Depending upon the joint where it is
placed, tendon can have different anatomic shapes [1]. Investigation on tendon has been carried out in various aspects [2-6] such as understanding the shape, structure, mechanical properties, tissue repair
and structure-function relationship. Like tendon, articular cartilage is also a soft
connective tissue, which covers the end surfaces of bones in synovial joints to distribute
compressive loading. While type I collagen fibrils are commonly found in tendon as
the highly organized and uniform fiber bundles, type II collagen fibrils are found
maximally in articular cartilage that are organized in a depth-dependent structure
[7-11], where the orientation of the local fibrils divides the cartilage depth into three
sub-tissue zones, namely superficial (fibrils parallel to tissue's surface), transitional
(random fibril orientation) and radial zones (fibrils perpendicular to the surface)
(Figure 1a).

Structural and biochemical alteration in the microstructure and composition of molecular
networks in the tissue due to any damage/degradation will eventually produce the clinical
symptoms of osteoarthritis. Till date, osteoarthritis cannot be diagnosed at its earliest
stage before the appearance of any clinical symptom. Change in biochemical constituents
indicates the tissue degradation in advance. Recent studies of cartilage using Fourier-Transform
Infrared Imaging (FTIRI) [12-19] show that infrared techniques with imaging capability have the potential to provide
quantitative information about chemical composition of tissue in its native and degraded
states. Since tendon has well-organized collagen structure, studies using infrared
technique have also been initiated on tendon [20-22]. Following the earlier research on FTIRI of tendon and cartilage [20], efforts have been made to understand the infrared anisotropy of articular cartilage
[23,24] for the tissue sections that consist of all the three zones (termed the regular section
in this article, Figure 1a). In these anisotropy studies, multiple infrared imaging data were acquired, each
for a different infrared polarization state in the 0°-180° angular space. Subsequent
analysis using all images can provide detailed information regarding the fibril orientation
and bond directions in cartilage [23,24]. When the long axis of the fibrils is in the plane of the tissue section, the bond
directions of amide I and amide II are approximately perpendicular and parallel to
the fibril axis respectively (Figure 1b1).

Since the matrix of collagen fibrils in articular cartilage has a unique three-dimensional
(3D) structure, different surfaces of a tissue block should have the fibrils in different
orientations, as illustrated in Figure 1a. In particular, we are interested in the infrared anisotropy when the long axis of
the collagen fibrils is perpendicular to the plane of a tissue section. In such a
case, a simple interpretation of the bond directions illustrated in Figure 1b1 would suggest a 'dot' for the amide II bond direction (Figure 1b2). In this study, the anisotropy of tendon and cartilage from all different surfaces
of the 3D tissue cube were investigated using infrared imaging. To the best of our
knowledge, there has been no study in literature regarding the infrared anisotropy
in the regular/parallel/cross sections of tendon and cartilage. Since the infrared
absorption of the amide I and amide II bonds has been found to have distinct anisotropy
in articular cartilage [23,24], this article focuses on the features of these two amide components in all sections.
(Since amide II and amide III bond directions are parallel, their anisotropy profiles
are of similar pattern, whereas the sugar component of proteoglycan has no anisotropy
[23].)

Methods

Sample preparation

Tendon and humeral head from mature canine, sacrificed for unrelated experiments,
were used in this study. Fresh canine achilles tendon was cleaned and freed of fat,
muscle and sheaths. Unfixed fragments of 10 mm long and 6 mm thick were cut, embedded
in OCT compound (cryo-embedding medium) and snap frozen using liquid nitrogen. Special
care was exercised in orienting the specimen parallel to the longest axis of the tendon.
The specimen blocks were sealed in aluminum foil and stored at -80°C until use. Rectangular
blocks of full thickness of cartilage attached to the underlying bone were harvested
from the central load-bearing region of the humeral head. To monitor the influence
of topographical variations, special attention was paid to the cartilage's location
and orientation on the joint surface by preserving the interface between the soft
tissue and the bone. The cartilage tissue blocks were placed in phosphate buffered
saline (pH 7.3) to prevent drying and were refrigerated until use. Standard histology
procedures were used to treat the cartilage tissue blocks, including overnight chemical
fixation with formol-cetylpyridiniumchloride (CPC), decalcification with 10% ethylene
diamine tetra acetic acid (EDTA)/Tris buffer for 7–10 days, and paraffin embedding
in tissue processor (RMC PTP 1530). (The infrared spectrum of paraffin does not interfere
with the cartilage spectra.)

Using a microtome (Micron HM325, Thermo Fisher Scientific, Waltham, MA), thin sections
(~6 μm thick) were cut from different surfaces of tendons as well as cartilage tissue
blocks and named as the regular, cross and parallel sections (Figure 1a). For articular cartilage, the regular sections contain all three zones of the tissue,
with the long axis of the fibrils in the superficial and radial zones in the plane
of the tissue section. The parallel sections of articular cartilage were acquired
at different tissue depths. Hence, while the parallel sections from the superficial
zone have the fibril in the plane of the sections, the parallel sections from the
radial zone have the long axis of the fibrils perpendicular to the plane of the sections
(cf Figure 1). Preserving the relative orientations among all parallel sections of cartilage is
also critically important. For tendon, the long axis of the specimen is parallel to
the long axis of the block; consequently, the regular and parallel sections of tendon
have the fibrils running parallel in the plane of the sections. The cross sections
of the tendon, however, only contain the 'ends' of the fibrils that are cut across,
similar to the case of cartilage's parallel sections from the radial zone. These sections
were placed on barium fluoride (BaF2) window as well as on commercially available mid infrared reflection study substrates
called MirrIR slides (Kevley Technologies, Chesterland, Ohio) to conduct FTIRI experiments.

Instrumentation details

Infrared images were acquired using a Spotlight 300 infrared imager from PerkinElmer
(Wellesley, Massachusetts). The apparatus consists of a FTIR spectrophotometer and
an infrared microscope. Liquid nitrogen cooled sixteen-element MCT (Mercuric Cadmium
Telluride) detector with a moving stage for scanning the sample constitutes the microscope.
The microscope also has a visible light source to focus the sample and to choose the
region of interest for data acquisition. The sections fixed on the mechanical stage
were undisturbed over the entire period of data collection. Experimental parameters
were unaltered for the entire set of experiments.

To investigate the anisotropy, a commercial wire grid infrared polarizer from PerkinElmer
was inserted between the sample and the detector (and will be referred as "analyzer" from now onwards). For each tissue section, infrared imaging experiments were repeated
thirteen times with the identical parameters except a 15° increment of the analyzer's
angle in the 0° – 180° angular space. For the analyzer angle 0°, the long axis of
the collagen fibrils is parallel to the x-axis of the x-y moving stage of the Infrared
Imager [23]. Transmission and reflection experiments were carried out for a selected region of
interest on each tissue section with a pixel size of 6.25 μm2. The spectral resolution of the instrument is 16 cm-1with data interval 8 cm-1 and 2 scans per pixel. Two to three identical sections were investigated in each type
of experiment; the results were highly consistent. Other experimental details can
be found elsewhere [23,24].

Data analysis

Each single infrared imaging experiment produces a 3D data cube, two spatial dimensions
and one spectral dimension in the mid infrared region (4000-750 cm-1). From this data cube, it is possible to extract two-dimensional (2D) chemi-maps
for any desired spectral interval. It is also possible to examine the infrared spectrum
at any spatial location of the tissue section. Since the previous studies have established
the anisotropy profile for amide I and amide II components of articular cartilage
in the spectral range 2000-1000 cm-1, this investigation also explored this spectral region. The baseline corrected chemi-maps
were extracted for amide I and amide II from the spectral range 1700 to 1600 cm-1 and 1600 to 1500 cm-1 respectively, from all infrared images. In the case of tendon, eight by eight pixels
in the chemi-maps were averaged to analyze the anisotropy at different surfaces of
the block for both amide I and amide II. Similar averaging was done for the parallel
sections of cartilage. For the regular sections of cartilage, eight consecutive columns
were averaged into one full-depth column so as to preserve the depth resolution of
the cartilage at 6.25 μm. Identical experimental parameters and data analysis approach
were used for all tissue sections from three different specimens, which in turn yielded
consistent results.

Results

Figure 2 shows the visible images of tendon and cartilage sections from different surfaces
of the tissue block. It is evident that the regular and parallel sections of tendon
have similar fibril morphology, with the tendon fibrils running parallel in the plane
of the tissue section. In contrast, the cross section of tendon has very different
morphology. For articular cartilage, the regular section contains three typical histological
zones; whereas each parallel section of cartilage has a very different fibril orientation,
depending upon the depth at which the section is obtained.

Tendon results

Figure 3 depicts the absorption anisotropy of amide I and amide II in tendon's regular, parallel
and cross sections. Two features can be observed. First, the absorption anisotropy
of amide I is stronger than that of amide II, which is due to greater bond strength
(double bond) of amide I whereas amide II absorption is caused by lesser bond strength
(single bond). Second, the anisotropy of amide I absorption is opposite to that of
amide II for all three sections, that ensures the perpendicularity of transition moment
directions of these amide bonds. For the parallel and regular sections of tendon,
since the fibril's long axis is parallel to the x-axis of the moving stage in both
orientations, their infrared anisotropy is similar to that of the radial zone fibrils
in regular sections of articular cartilage (the amide I anisotropy has a maximum at
0° and a minimum at 90°; and the same for amide II is opposite [23,24]). An interesting result is the amide anisotropy in the cross sections – though the
anisotropy is weaker compared to the same in other two surfaces, the angular dependency
remains the same.

Figure 3.Absorption anisotropy of amide I (a) and amide II (b) of tendon in the regular, parallel
and cross sections.

To further investigate the anisotropy of the cross sections from tendon, the same
cross section was placed at three different orientations (θ = 0°, ~65° and 90°) with
respect to the polarization axis and the anisotropy experiments were repeated at these
three orientations. Figure 4 shows the anisotropy profiles of amide I and amide II for these three orientations.
It is clear that both amide vibrations have distinct anisotropy with the perpendicularity
between them, even though the cross sections of the tendon do not have a visible fibril
arrangement (cf Figure 2a). This result has two implications. First, the schematic assumption for the amide
II orientation as illustrated in Figure 1b2 needs further investigation (see later in Discussion). Second, these amide bonds
have a fixed orientation in the tissue's cross section with respect to the local fibril
structure. (These experiments were conducted on various cross sections and the results
are found to be consistent.)

Figure 4.Absorption anisotropy of amide I (a) and amide II (b) of tendon's cross section at
three different sample orientations.

Another noticeable feature in Figure 4 is the 'phase shift' in the minimum and maximum absorption locations (angles) for
a sample that is not oriented parallel/perpendicular with respect to the analyzer
0°. Though it appears like a full cycle in 0–180° angular space, the difference between
the minimum and maximum absorption will always be 90°. To verify this observation,
the regular section of the tendon was imaged when the section was tilted by about
~60° with respect to the initial orientation used in Figure 3. The results are shown in Figure 5, where both profiles of the amide anisotropy from this regular section show the 'phase
shift'. (i.e., the amide I plot in Figure 5 is 'phase shifted' from the amide I plot in Figure 3a.) This anisotropy shift illustrates the importance of the specimen orientation in
the FTIRI anisotropy experiment, as the anisotropy is a polarization dependent phenomenon.

Figure 5.The phase shift in the absorption anisotropy due to a sample rotation (the same tendon
section as in Figure 3 now oriented at ~60°).

Cartilage results

Based on the results of tendon, investigations are made on the anisotropy of cartilage
for both regular sections as well as the parallel sections obtained at different zones.
The results of the regular sections (that contain all three histological zones) are
identical to our previously published data [23,24]. Since the fibrils are in the plane of the cartilage's regular sections, which is
similar to the fibril orientation of tendon's regular/parallel sections, the amide
anisotropy in these cartilage sections is identical to those in the tendon's regular/parallel
sections (cf Figure 3). The only additional feature in the cartilage case is the perpendicular nature the
fibril orientation between the superficial and radial zones of the tissue (cf Figure
1a), which causes the infrared anisotropy of the same amide component to be opposite
between the two zones.

Since the regular section anisotropy is well-established, parallel sections of cartilage
is focused in this article. Figure 6 shows the infrared anisotropy profiles at the superficial and radial zones of cartilage
parallel sections respectively. In the superficial zone (Figure 6a), the anisotropy of amide I is opposite to that of amide II, which is the same as
in regular section of cartilage. A unique feature of the infrared anisotropy in cartilage
is its depth dependency. In regular sections of cartilage, the anisotropy of both
amide components decreases gradually from the superficial zone to the transitional
zone and increases in opposite direction gradually from the transitional zone to the
radial zone. In this study where each parallel section has a 6-μm separation from
the next one, the same trend in infrared anisotropy is observed. The two plots of
amide I and amide II (Figure 6a) are from two parallel sections, separated by a 42 μm gap in between. The deeper
section has the same but weaker anisotropy.

The parallel sections from the radial zone, however, have a different anisotropy.
Figure 6b shows that, while amide I retains a distinct anisotropy, the amide II anisotropy
in the radial zone becomes very weak. For this type of canine cartilage, the transitional
zone has been found approximately from 70 μm to 120 μm [25]. From about 250 μm onwards, the tissue is well into its radial zone where all fibrils
are expected to be parallel to each other (cf Figure 1a). Consequently, all parallel sections in the deep radial zone are expected to have
the same anisotropy. This is true since there is little variation between the two
plots of each amide component in Figure 6b, even the two parallel sections are 48 μm apart. However, the observation of amide
I anisotropy in the radial zone's parallel sections was not expected, if one considers
the schematic illustrations in Figure 1.

Discussion

In infrared polarization experiments with cartilage/tendon, maximum and minimum absorption
occurs when the polarization axis is parallel and perpendicular to amide bond transition
moment directions respectively. Our previous results have verified such anisotropy
for both amide I and amide II components using the regular sections of cartilage,
as illustrated in Figure 1b1. To investigate infrared anisotropy for the tissue sections where the long axis of
the fibrils is perpendicular to the section plane, such simple illustration is not
sufficient. Hence, a detailed illustration is given in Figure 7, which incorporates the tilting angles of the transitional moments of amide bonds
in collagen fibrils as well as the effect of polarization in infrared imaging.

Figure 7.(a) The transitional moments of one pair of amide bonds at one location in the triple
helix. The distribution of numerous amide bonds along the fibril axis would be similar to
the cone structures as in (b). When the long axis of the fibrils is parallel to tissue
section (b), the 'projection' of the transition moment 'cone' varies its size at the
2D z-z' plane with the change of polarization state. Consequently, there will be infrared
anisotropy in (b). When the long axis of the fibrils is perpendicular to the tissue
section (c), the 'projection' of the transition moment 'cone' remains the same at
the 2D z' plane regardless of the polarization state. Consequently, there will be
no infrared anisotropy in (c).

It is well known in literature that the transition moments of amide I and amide II
have tilting angles with respect to the axis of the alpha-helix [26], as shown in Figure 7a. Since the amide bonds are fixed in the peptide chains and the fibril contains three
identical chains in a triple helix, it is evident that the transitional moments of
amide vibrations also spiral around the long axis of the fibrils. Hence, there exist
two situations when performing infrared polarization experiment: (a) when the long
axis of the fibril is in the plane of the tissue section (Figure 7b), which is the case of regular/cross sections of cartilage with all three zones in
the plane as well as the case of regular/parallel sections of tendon, and (b) when
the long axis of the fibril is perpendicular to the plane of the tissue section (Figure
7c), which is the case of parallel section of cartilage in the radial zone as well as
the cross sections of tendon.

When one carries out a polarization experiment, the polarization axis is alwaysrotated in the plane of the tissue section. In situation (a), the distribution of
an amide bond around the numerous fibrils will have the shape of a cone, as shown
in Figure 7b. When the polarization axis is rotated in the tissue plane (the z-z' plane), the
transition moment vector of an amide component varies as the function of the polarization
state, thus yielding anisotropy. In contrast, in situation (b), the infrared irradiation
is polarized in the 2D z' plane, where the resultant bond vectors from the cone would
not change as the function of the polarization state (Figure 7c), thus yielding no anisotropy.

In the study of tendon's cross sections, both amide I and amide II showed strong anisotropy
(Figure 3). The result of cartilage's parallel sections from the radial zone, amide I has strong
anisotropy but amide II is nearly isotropic (Figure 6b). The tendon results from the cross sections suggest that the long axis of the fibers
is not perpendicular to the plane of the tissue section. This can be explained by
a well known wavy/zigzag nature of collagen bundles in tendon [1,2], which may result in a residual anisotropy in tendon's cross sections observed in
this study. In comparison, the isotropic nature of the amide II component in the parallel
sections of cartilage's radial zone implies that the long axis of the fibrils in the
radial zone of the cartilage is indeed perpendicular to the plane of the tissue section
(the situation (b) discussed above). However, the same situation (b) cannot explain
the observed anisotropy of amide I in the radial zone of cartilage. This conundrum
might be, in part, due to the small concentration of connecting fibrils in cartilage's
radial zone [27]. Further experiments have been planned to investigate the nature of the 3D fibril
orientation in articular cartilage.

Conclusion

The infrared anisotropy of tendon as well as cartilage has been investigated in regular,
parallel and cross sections from a 3D tissue block. The results are mutually consistent,
when the long axis of the fibrils is parallel to the plane of the tissue section.
An interesting situation is when the long axis of the fibrils is perpendicular to
the plane of the tissue section. Though the infrared anisotropy of amide components
in tendon cross sections would be expected to be isotropic, the experimental results
show a clear anisotropy for both amide I and amide II components in tendon. This could
be attributed to the zigzag nature of the collagen fibers in tendon. The results of
the parallel sections from cartilage's superficial zone are similar to that of regular
sections in cartilage, which is also comparable to the anisotropy in tendon's regular
and parallel sections. The parallel sections from cartilage's radial zone have a nearly
isotropic amide II absorption and a distinct amide I anisotropy. The origins of these
features are investigated with the aid of a schematic model.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

NR carried out the FT-IR imaging experiments, analyzed the raw data and drafted the
manuscript. YX conceived of the study, participated in its design and coordination,
finalized the data analysis, interpreted the concluding results, made the final figures,
revised and completed the manuscript. AB performed the histological sectioning of
the tissue blocks and participated in the experiments. All authors read and approved
the final manuscript.